ES2625501T3 - Improved procedure for the production of cellulolytic and / or hemicellulolytic enzymes - Google Patents

Abstract

Method of production of cellulolytic and / or hemicellulolytic enzymes by a cellulolytic and / or hemicellulolytic microorganism comprising at least one growth phase in the presence of a carbon source and at least one production phase in the presence of an inducing substrate, in wherein said inducing substrate is a mixture of glucose or cellulosic hydrolysates, lactose and xylose, or a solution of hemicellulolytic hydrolysates, the content of each of the constituents of the mixture defined by the following limits being: - from 40 to 65% by weight of glucose, - from 21 to 25% by weight of lactose and - from 10 to 39% by weight of xylose, the sum of these three constituents being 100%, and the microorganism belongs to the species Trichoderma reesei , and is deleted for catabolic repression by glucose.

Description

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

DESCRIPTION

Improved process for the production of cellulolytic and / or hemicellulolytic enzymes Scope of the invention

The invention is related to a process for the production of enzymes for the hydrolysis of lignocellulosic biomass. Prior art

The increase in bioethanol production capacities due to its biofuel qualities is today a current issue. The incorporation objectives are being discussed within the European Union on the basis of an initial proposal of a 20% use of renewable energies from 2020 to 10% incorporation of biofuels according to criteria of duration that should favor to second generation biofuels produced from lignocellulosic biomass.

Lignocellulosic biomass is characterized by a complex structure consisting of three main poftmeros: cellulose, hemicellulose and lignin.

Classically, the process of transforming biomass into ethanol comprises several stages. A pretreatment makes cellulose and eventually hemicelluloses accessible, which are the objectives of enzymatic hydrolysis to enzymes. The goal of pretreatment is to modify the physical and physicochemical properties of lignocellulosic material, in order to improve the accessibility of cellulose trapped in the lignin and hemicellulose matrix. The enzymatic hydrolysis stage allows the transformation of cellulose and hemicelluloses into sugars through the use of cellulolytic and / or hemicellulolytic enzymes.

The sugars obtained by hydrolysis of lignocellulosic biomass are pentoses (mainly xylose and arabinose), disacarids (cellobiose) and glucose, which can be fermented by microorganisms. Glucose can, for example, be easily transformed into ethanol by Saccharomyces cerevisiae yeast during the alcoholic fermentation stage.

Finally, a distillation step allows the product from the fermentation to be separated and recovered, thus obtaining the ethanol in the previous case, from the fermentation must.

The different technical-economic studies demonstrate the need to reduce the cost related to the stage of enzymatic hydrolysis to bring the cost of ethanol produced to values close to those of ethanol obtained from starch.

At the present time, industrial cellulases are mainly produced by a filamentous fungus, Trichoderma reesei, thanks to its strong cellulase secretion power.

One of the means for reducing costs is to optimize the operating conditions of the cellulase production process so as to increase productivity, or to obtain an enzymatic cocktail that has an improved specific activity.

Wild strains of Trichoderma reesei have the power to secrete, in the presence of an inducing substrate, an enzymatic complex well adapted for cellulose hydrolysis. Enzymes in the enzyme complex contain three main types of activity: endoglucanases, exoglucanases and cellobiases. Other proteins, such as xylanases, necessary for the hydrolysis of lignocellulosic biomass, are also produced by Trichoderma reesei. The presence of an inducing substrate is indispensable for the expression of cellulolytic and / or hemicellulolytic enzymes.

The regulation of cellulase genes on different carbon sources has been studied in detail. Glucose exerts a catabolic repression on the production of cellulases. This is induced in the presence of cellulose, of its hydrolysis products, such as cellobiose or certain oligosaccharides, particularly disacarids, such as lactose or soforose (llmen and al. 1997, Appl. Environ. Microbiol. Vol. 63, pages 1298 -1306). The nature of the carbon substrate has a strong influence on the composition of the enzyme complex. Thus, xylose, associated with an inductive carbon substrate such as cellulose or lactose, allows to significantly improve the activity of xylanase when present at limiting concentrations of the order of between 0.5 and 1 mM (Mach-Aigner et al., 2010 Applied and Environmental Microbiology, Vol. 76 No. 6, pages 1770-1776). The solubilization of residual hemicelluloses (xylanes) by xylanases allowed to favor enzymatic hydrolysis (Varnai et al., 2010 Enzyme and Microbial Technology Vol. 46 pages. 185-193).

In order to obtain good enzymatic productivity, it is necessary to provide a rapidly assimilable carbon source to allow rapid growth of Trichoderma reesei, and an inducing substrate that allows the expression of cellulases and their secretion in the culture medium. Cellulose can play these two roles. However, it is

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

Difficult to use on an industrial scale, and has been replaced by soluble carbon sources, such as lactose, which also plays the role of inducing substrate.

Other sugars such as cellobiose or soforose have also been described as inducers (limen and al. (1997), Foreman and al. (2003) Biol Chem 278, pages 31988-31997, Pakula and al. (2005) Microbiology, 151, pages 135143) but they are too expensive to be used on an industrial scale.

In general, it has been found that cellulose productions by Trichoderma reesei with soluble substrates are much lower than those obtained with cellulose in "batch" mode (that is, in a closed medium) due to the repressive effect of sugars easily assimilable to a high concentration.

Patent FR-B-2 555 603, filed in the name of the Claimant, proposes a continuous feeding of the carbonized substrates that allows to increase the catabolic repression by limiting the residual concentration in the crops and optimizing the amount of sugars, to obtain a better yield and better enzymatic productivity. The procedure described in that patent proposes to start the feeding with substrate using soluble sugars as a carbon source, possibly in the form of a mixture. However, a continuous limiting contribution of glucose or xylose not associated with lactose or another cellulase inducer (soforose, cellobiose, ...) does not allow obtaining large enzyme productivity.

Lactose remains, in the industrial production processes of cellulolytic enzymes, one of the most appropriate substrates. In addition to being high, its price is very valuable and represents approximately one third of the cost price of enzymes.

One of the solutions contemplated and presented in EP-B-0 448 430 consists in the use of carbon substrates from the sector, for example, hydrolyzed hemicelluloses, as a source of inductive carbon. However, productivity remains below approximately 50% with respect to the procedure that uses lactose as the only inducing substrate.

Patent application WO 2009/026716 describes a process for the production of cellulases in which hemicellulolytic derivatives represent more than 40% of the carbon source, and the sugars that induce cellulose production represent between 3 and 20% of this mixture. This procedure allows the production of at least twice more cellulases with respect to a procedure that uses only two sugars from hemicelluloses. However, the production yields of cellulases described are still lower than those of a process that uses only lactose.

Current research shows that it is necessary, to improve the production processes of cellulolytic and / or hemicellulolytic enzymes, to develop new inducing substrates, which behave at least like those that use lactose, and with a lower cost.

It is in this area that the present invention is inscribed.

Summary of the invention

The present invention describes a process for producing cellulolytic and / or hemicellulolytic enzymes in which the inducing substrate is a mixture of glucose, lactose and xylose.

Detailed description of the invention

The process of production of cellulolytic and / or hemicellulolytic enzymes by a cellulolytic and / or hemicellulolytic microorganism according to the present invention comprises at least one growth phase in the presence of a carbon source and at least one production phase in the presence of a inducing substrate, wherein said inducing substrate is a mixture of glucose or cellulosic hydrolysates, lactose and xylose, or a solution of hemicellulolytic hydrolysates, the content of each of the constituents of the mixture being defined by the following limits :

- 40 to 65% by weight glucose,

- from 21 to 25% by weight of lactose and -of 10 to 39% by weight of xylose,

the sum of these three constituents being equal to 100%.

The inducing substrate is devoid of any other sugar than the constituents indicated above. Thus, the relative proportions of each of the constituents are chosen so that the sum of the weight contents is equal to 100%.

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

Thanks to the process according to the invention, which uses a mixture of particular sugars as an inducing substrate, the final concentrations of proteins obtained are between 50 and 60% higher than those obtained with the lactose used as the only production substrate. They are also approximately 3 times higher than those obtained with the improved mixtures rich in xylases such as those described in the prior art document WO 2009/026716.

The method according to the present invention uses a glucose based mixture. More than 60% of the lactose, inducer of cellulose production, can be replaced by glucose, therefore known to be a repressor of cellulose production, with an improved behavior of the inductive mixture. Glucose has a much lower cost than lactose, and a water solubility approximately 3 times higher. Therefore, it is possible to decrease the volumes involved. Thus, in this procedure, glucose is used at the same time in the growth phase and also in the production phase in an amount of 60% of the mixture constituting the inducing substrate.

The glucose used in the sugar mixture can also be replaced by glucose from the enzymatic hydrolysis stage of cellulose, therefore coming directly from the process of transforming lignocellulosic biomass into ethanol. This helps to reduce the cost of enzyme production by using the process co-products. We speak therefore of cellulose hydrolysates.

The xylose used in the mixture that constitutes the sugar inducer can be replaced by a solution of hemicellulolytic hydrolysates, derived from the process of transforming lignocellulosic biomass into ethanol, and coming particularly from the pretreatment of biomass.

On the other hand, the possibility of using a very concentrated mixture of sugars allows advantageously to limit the risks of contamination.

The fact of adding xylose to the mixture that constitutes the inducing substrate makes it possible to significantly increase the xylanase activity of the final enzyme cocktail. The xylose can be advantageously substituted by a solution of hemicellulosic hydrolyzate, derived from the hydrolysis of the hemicelluloses during the pretreatment of lignocellulosic biomass in the production processes of second generation biofuels, and which may be concentrated if necessary.

In a preferred manner, the mixture constituting the inducing substrate comprises between 50 and 65% by weight of glucose, between 22 and 24% by weight of lactose and between 15 and 25% by weight of xylose, possibly from a pretreatment of the lignocellulosic biomass, the sum of the constituents being equal to 100%.

Most preferably, the inducing substrate is a mixture consisting of 60% by weight of glucose, 23% by weight of lactose and 17% by weight of xylose.

The carbon substrate used during the growth phase is chosen from glucose, xylose, lactose, residues obtained after ethanol fermentation of monomeric sugars from enzymatic hydrolysates of cellulosic biomass and / or a crude extract of water-soluble pentose from eventually pretreatment of a cellulosic biomass.

Most preferably, the growth substrate is glucose.

The industrial strains used belong to the species Trichoderma reesei, modified to improve the cellulolytic and / or hemicellulolytic enzymes through mutation-selection processes. For example, the IFP strain CL847 will be mentioned. Improved strains can also be used by genetic recombination techniques. They must be deleted for catabolic repression by glucose (A CRE1), such as CL847.

These strains are grown in agitated and aerated bioreactors in conditions compatible with their growth and the production of enzymes. The conditions are such that the pH is set between 3.5 and 6, and the temperature between 20 and 35 ° C. A pH of 4.8 and a temperature of 27 ° C will preferably be chosen during the growth phase, and a pH of 4 and a temperature of 25 ° C during the production phase. The aeration rate, expressed in volume of air per volume of reaction medium and per minute or vvm applied during the procedure, is between 0.3 and 1.5 min-1, and the rotation speed or rpm should allow to regulate the pressure of O2 to between 20% and 60%. Preferably, an aeration of 0.5 vvm and agitation will be chosen that allows the pressure of 30% O2 to be regulated.

Depending on its nature, the carbon substrate chosen for obtaining the biomass is introduced into the bioreactor before sterilization, or is sterilized separately and then introduced into the bioreactor after sterilization of the latter, to obtain an initial concentration of sugars between 15 and 60 g / l.

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

With respect to the production phase, an aqueous solution is prepared containing the inducing substrate consisting of the mixture of glucose / lactose / xylose or a solution of hemicellulosic hydrolyzate chosen for the production phase of the enzymes at a concentration of between 350 and 600 g / l in the feeding solution used.

Preferably, the concentration is between 450 and 550 g / l.

The aqueous solution is injected after depletion of the initial substrate, so that an optimized amount is provided. The flow of introduction of the feeding solution is between 30 and 45 mg per gram of cells and per hour.

The residual concentration of sugar in the culture medium is less than 1 g / l during the production phase ("fed-batch"), so that biomass production is limited. Preferably, this concentration is less than 0.5 g / l, and even more preferably 0.1 g / l.

Examples

Among the following examples, Example 1 presents a culture that uses glucose as a carbon substrate during the growth phase and the production phase. This example shows the low production of proteins obtained when this sugar is used as the only carbon substrate, even if the flow is limiting during the fed-batch phase (residual glucose concentration close to 0). The second example represents the reference fermentation through the use of lactose as a carbon substrate during the growth phase and the production phase, which allows an important production of proteins. Example 3 reproduces an experiment that uses a mixture such as that described in patent application WO 2009/026716 that contains a high proportion of xylose that, according to the authors, greatly increases the production of cellulases with respect to an experiment in which xylose or hemicellulosic derivatives are used as unique carbon substrates. Examples 4 to 6 are of patents conforming to the process according to the present invention.

Example 1: production of enzymes with glucose (not in accordance with the invention)

The production of cellulases is carried out in a mechanically agitated bioreactor. The mineral medium has the following composition: KOH 1.66 g / l, H3PO4 85% 2 ml / l, (NH4) 2SO4 2.8 g / l, MgSO4 ■ 7 H2O 0.6 g / l, CaCl2 0, 6 g / l, MnSO4 3.2 mg / l, ZnSO4 ■ 7 H2O 2.8 mg / l, CoCI2 10 4.0 mg / l, FeSO4 ■ 7 H2O 10 mg / l, Corn Steep 1.2 g / l , defoamer 0.5 ml / l.

The bioreactor containing the mineral medium is sterilized at 120 ° C for 20 minutes, the carbonated glucose source is sterilized separately at 120 ° C for 20 minutes and then sterilely added to the bioreactor, so that a final concentration of 30 g / l The bioreactor is grown at 10% (v / v) with a liquid preculture of the Trichoderma reesei CL847 strain. The preculture mineral medium is identical to that of the bioreactor except for the addition of potassium phthalate to 5 gl-1 to buffer the pH. The growth of the preculture fungus is carried out using glucose as a carbon substrate, at a concentration of 30 g / l. The growth of the inoculum lasts between 2 and 3 days and is carried out at 28 ° C in a stirred incubation oven. The transfer to the bioreactor is carried out when the residual glucose concentration is less than 15 g / l.

The experiment conducted in the bioreactor comprises two phases:

- a growth phase in the carbon-carbon substrate (initial concentration = 30 g / l) at a temperature of 27 ° C and a pH of 4.8 (regulated by 5.5 M ammonia). The aeration is 0.5 vvm and the stirring is increased up to between 200 and 800 rpm depending on the pO2 (the dissolved oxygen pressure), which is maintained above 30%.

- a phase of enzyme production. When the initial substrate of the fermenter has run out and the glucose solution has been injected at 250 g / l continuously with a flow rate of between 30 and 40 mg per g of cells and per hour up to 164 hours. The temperature is reduced to 25 ° C and the pH to 4 until the end of the culture. The pH is regulated by the addition of a 5.5 N ammonia solution that provides the nitrogen necessary for the synthesis of excreted proteins. The dissolved oxygen content is maintained above 15-20% by aeration and stirring.

Enzyme production is followed by the addition of extracellular proteins by the method of Lowry and standard BSA, after separation of the mycelium by filtration or centrifugation. The cellulological activities determined are:

- the filter paper activity (UPF: filter paper unit) that allows to calculate the overall activity of the enzymatic set of endoglucanases and exoglucanases

- the activities of aryl p-glucosidase and xylanases for specific activities.

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

UPF activity is measured with a Whatman paper No. 1 (procedure recommended by the IUPAC biotechnology commission) at an initial concentration of 50 g / l; The test sample of the enzymatic solution to be analyzed is determined, which releases the equivalent of 2 g / l glucose (colorimetric analysis) in 60 minutes. The principle of filter paper activity is to determine the amount of reduced sugars from a Whatman paper No. 1 (procedure recommended by the IUPAC biotechnology commission) by analyzing DNS (dinitrosalidic acid).

The substrate used to determine the activity of aryl p-glucosidase is p-nitrophenyl-p-D-glucopyranoside (PNPG). It is cleaved by p-glucosidase, which releases p-nitrophenol.

A unit of aryl p-glucosidase activity is defined as the amount of enzyme needed to reduce 1 pmol of p-nitrophenol from PNPG per minute, and is expressed in 1U / ml. The principle of the analysis of xylanase activity is based on the determination by DNS analysis of the amount of reduced sugars from the hydrolyzed xylanase solution. This method of analysis uses the reducing properties of sugars, and mainly of xylose. The xylanase activity is expressed in 1U / ml, which corresponds to the amount of enzyme needed to produce 1 pmol of xylose per minute.

The specific activities are obtained by dividing the activities expressed in lU / ml by the concentration of proteins. They are expressed in lU / mg.

The analytical determinations on the final must of Example 1 provide the following results:

The analytical determinations on the final must provide the following results:

Biomass g / l 15.2

Protemes g / l 2.9

UPF lU / ml 1.4

Xylanase 29.1 lU / mg

Specific p-Glucosidase 0.35 lU / mg

The CL847 strain is depressed against the catabolic repression exerted by glucose on cellulase production (the CRE1 gene has been deleted). It seems that even for a fed-batch carried out under conditions of glucose limitation, the final production of proteins is very low.

Example 2: production of enzymes with lactose (not in accordance with the invention)

Enzyme production is carried out under the same conditions as in Example 1. The carbon substrate during the growth and production phases is pure lactose. Lactose is an important inducer of cellulase production. This is the substrate most used industrially for the production of cellulases.

After 30 hours of growth, after depletion of the initial substrate, the fed-batch solution at 250 g / l is injected continuously with a flow rate of 35 mg per g of cells and per hour up to 164 hours.

The analytical determinations on the final must provide the following results:

Biomass g / l 13.5

Protemes g / l 37.8

UPF lU / ml 22.1

Xylanase 408.5 lU / ml

Specific p-Glucosidase 0.96 lU / mg

Example 3: production with the mixture of 97% xylose / 3% of sugars inducing the production of cellulases or CIC (not in accordance with the invention)

The experiment was carried out under the same conditions as in Example 1 by the use of pure xylose as the only carbon substrate during the growth phase, and in the production phase, the mixture of 97% xylose / 3% CIC filed in patent application Wo 2009/026716 A1. CICs (sugars that induce cellulose production) are a mixture of sugars that allows the induction of cellulase production. Its composition is as follows: 56% gentiobiosa, 14% soforosa, 10% trehalose, 6% cellobiose, 6% maltotriose, 8% glucose.

After depletion of the initial substrate, the mixture of 97% xylose / 3% CIC at 360 g / l is injected according to the recommendations described, with a flow rate of 0.4 g of carbon L-1h-1

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

The analytical determinations on the final must provide the following results:

Biomass 14.3 g / l

Protemes 18.7 g / l

UPF 6.4 lU / ml

Xylanase 6000.1 lU / ml

Specific UPF 0.34 lU / mg

Specific p-Glucosidase 1.15 lU / mg

The final production of proteins is close to that presented in WO 2009/026716 A1, in which the authors have obtained a final concentration of proteins of 25 g / l with strain P59G. The specific activity of FPase (0.34 lU / mg) is low, while the activity of xylanase is very high.

Example 4: production with the mixture of lactose (23%) / glucose (60%) / xylose (17%) at 500 g / l (according to the invention)

The experiment of Example 4 is carried out under the same conditions as in Example 1 with glucose as a carbon growing substrate, at 60 g / l. Then we have used, during the production phase in "fed-batch" mode, a solution in which three carbonized substrates are mixed according to the following proportions: lactose (23%) / glucose (60%) / xylose ( 17%) at 500 g / l. This mixture has been injected at 45 mgg'1h'1. The analytical determinations on the final must provide the following results:

Biomass 26.1 g / l

Proteins 61.8 g / l

UPF 33.7 lU / ml

Xylanase 4017 lU / ml

Specific UPF 0.55 lU / mg

Specific p-Glucosidase 1.2 lU / mg

The final production of proteins is more than 3 times higher than in example 3. The filter paper activity, which is indicative of cellulase activity, is approximately 5 times higher.

The xylanase activity is 10 times higher than in Example 2 performed with lactose as the only carbon substrate for growth and production, but approximately 50% lower than in Example 3.

Example 5: use of the mixture of lactose (23%) / glucose (60%) / solution of C5 sugars (17%) at 500 g / l (according to the invention)

Example 5 is carried out under the same conditions as in example 4. Glucose is used during the growth phase at a concentration of 15 g / l. The xylose of the fed-batch solution is replaced by a soluble hemicellulosic solution or a solution of C5 sugars from wheat straw impregnated with 0.08 N sulfuric acid and pretreated with a steam explosion (19 bar, 5 minutes). The mixture is injected at 30 mgg'1h'1 for 170 h.

The analytical determinations on the final must provide the following results:

Biomass 19.1 g / l

Protemes 57.3 g / l

UPF 25.1 lU / ml

Xylanase 1151 lU / ml

Specific UPF 0.44 lU / mg

Specific p-Glucosidase 1.4 lU / mg

This experiment has allowed to increase the production of proteins by more than 50% with respect to example 2, and the activity of xylanase is almost 3 times higher. Most of the lactose is replaced during the growth and production phase by glucose and the C5 sugar solution. The glucose concentration during the growth phase has been reduced with respect to that of example 4, from 60 to 15 g / l. The biomass concentration has decreased from 26 to 19 g / l, but this has little impact on the final protein concentration (7% reduction). Thus, we have, therefore, an optimal use of sugars and a notable decrease in the cost of the carbon source.

Example 6: use of the mixture: lactose (23%) / C6 hydrolyzates (56%) / xylose (21%) at 500 g / l (according to the invention)

Example 6 is carried out under the same conditions as in example 4. Glucose is used during the growth phase at a concentration of 15 g / l. The glucose of the fed-batch solution is replaced by a solution of hydrolyzate from the enzymatic hydrolysis of wheat straw impregnated with sulfuric acid 0.08 N and pretreated

with a steam explosion (19 bar, 5 minutes). Enzymatic hydrolysis of wheat straw has been carried out at 50 ° C and at a pH of 4.8. It has resulted in a 95% hydrolysis of cellulose in glucose.

Lactose and xylose have been dissolved in this solution so that it is obtained in the mixture:

5

lactose (23%) / C6 hydrolyzates (56%) / xylose (21%) at 500 g / l

The mixture is injected at 30 mgg'1h'1 for 170 h during the fed-batch phase.

The analytical determinations on the final must provide the following results:

10

Biomass 16.1 g / l Proteins 43.1 g / l UPF 31.4 lU / ml Xylanase 6595.1 lU / ml 15 Specific UPF 0.73 lU / mg

Specific p-Glucosidase 1.2 lU / mg

The enzymatic cocktail obtained is of very good quality, since the final activity of FPase is close to the activity obtained in example 4, even if the protein concentration is lower. The final activity of FPase 20 is approximately 5 times higher than that obtained with Example 3, and approximately 50% higher than that obtained for Example 2, in which lactose is used as the only carbon substrate during the fed-batch phase. .

Claims (9)

5 10 fifteen twenty 25 30 35 40 1. Process for the production of cellulolytic and / or hemicellulolytic enzymes by a cellulolytic and / or hemicellulolytic microorganism comprising at least one growth phase in the presence of a carbon source and at least one production phase in the presence of an inducing substrate , wherein said inducing substrate is a mixture of glucose or cellulosic hydrolysates, lactose and xylose, or a solution of hemicellulolytic hydrolysates, the content of each of the constituents of the mixture being defined by the following limits: - 40 to 65% by weight glucose, - from 21 to 25% by weight of lactose and -of 10 to 39% by weight of xylose, the sum of these three constituents being equal to 100%, and The microorganism belongs to the species Trichoderma reesei, and is delegated for catabolic repression by glucose. 2. The method according to claim 1, wherein the contribution of the inductor substrate is carried out in solution, the concentration of the inductor substrate being in the feed solution used during the production phase of between 350 and 600 g / l. 3. Method according to claim 2 wherein the concentration is between 450 and 550 g / l. 4. Method according to one of the preceding claims wherein the mixture constituting the inducing substrate comprises 50 to 65% by weight of glucose, 22 to 24% by weight of lactose and 15 to 25% by weight of xylose, the sum of the constituents of the mixture being equal to 100%. 5. Method according to claim 4 wherein the inducing substrate is a mixture consisting of 60% by weight of glucose, 23% by weight of lactose and 17% by weight of xylose. Method according to one of the preceding claims in which the carbon substrate used during the growth phase is chosen from glucose, xylose, lactose, residues obtained after ethanol fermentation of the monomeric sugars of the enzymatic hydrolysates of Cellulosic biomass and / or a crude extract of water-soluble pentoses from the pretreatment of a cellulosic biomass. 7. Method according to one of the preceding claims wherein the pH is adjusted between 3.5 and 6 and the temperature is between 20 and 35 ° C. 8. Method according to one of the preceding claims wherein the flow of introduction of the feed solution is 30 to 45 mg per gram of cells and per hour. 9. Method according to one of the preceding claims wherein the residual concentration of sugar in the culture medium during the production phase is less than 1 g / l, preferably 0.5 g / l and most preferably 0.1 g / l.